The present invention relates to the production of adeno-associated virus in insect cells.
Viruses of the Parvoviridae family are small DNA animal viruses characterized by their ability to infect particular hosts, among other factors. Specifically, the family Parvoviridae is divided between two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. The subfamily Parvovirinae (members of which herein are referred to as the parvoviruses includes the genus Dependovirus, the members of which are unique in that, under most conditions, these viruses require coinfection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture. The genus Dependovirus includes adeno-associated virus (AAV), which normally infects humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). The parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, xe2x80x9cParvoviridae: The Viruses and Their Replication,xe2x80x9d Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).
In recent years, AAV has emerged as a preferred viral vector for gene therapy due to its ability to efficiently infect both nondividing and dividing cells, integrate into a single chromosomal site in the human genome, and pose relatively low pathogenic risk to humans. In view of these advantages, recombinant adeno-associated virus (rAAV) presently is being used in gene therapy clinical trials for hemophilia B, malignant melanoma, cystic fibrosis, and other diseases.
AAV is able to infect a number of mammalian cells. See, e.g., Tratschin et al., Mol. Cell Biol., 5(11):3251-3260 (1985) and Grimm et al., Hum. Gene Ther., 10(15):2445-2450 (1999). However, AAV transduction of human synovial fibroblasts is significantly more efficient than in similar murine cells, Jennings et al., Arthritis Res, 3:1 (2001), and the cellular tropicity of AAV differs among serotypes. See, e.g., Davidson et al., Proc. Natl. Acad. Sci. USA, 97(7):3428-3432 (2000) (discussing differences among AAV2, AAV4, and AAV5 with respect to mammalian CNS cell tropism and transduction efficiency). Most commonly, rAAV is produced in 293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines. See, e.g., U.S. Pat. Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, and 5,688,676; U.S. patent application 2002/0081721, and International Patent Applications WO 00/47757, WO 00/24916, and WO 96/17947. Although virus-like particles (VLPs) of parvoviruses have been produced in insect cells (see, e.g., Ruffing et al., J. Virol., 66(12):6922-6930 (1992), Brown et al., J. Virol., 65(5):2702-2706 (1991), and Yuan et al., Virology, 279(2):546-547 (2001)), the production of infectious AAV in nonmammalian, invertebrate cells currently is not known. The replication of parvoviral viral genomes, including, particularly, Dependovirus genomes, in nonmammalian, invertebrate cells, is similarly heretofore unknown.
The difficulties involved in scaling-up rAAV production for clinical trials and commercialization using current mammalian cell production systems can be significant, if not entirely prohibitive. For example, for certain clinical studies more than 1015 particles of rAAV may be required. To produce this number of rAAV particles, transfection and culture with approximately 1011 cultured human 293 cells, the equivalent of 5,000 175-cm2 flasks of cells, would be required. Related difficulties associated with the production of AAV using known mammalian cell lines are recognized in the art. See, e.g., Grimm et al, supra. There also is the possibility that a vector destined for clinical use produced in a mammalian cell culture will be contaminated with undesirable, perhaps pathogenic, material present in a mammalian cell.
In view of these and other issues there remains a need for alternative and improved methods of efficiently, safely, and economically producing a large amount of infectious rAAV particles. The invention provides such methods. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The present invention provides a method of producing AAV in an insect cell. The method comprises providing at least one insect cell-compatible vector. The vector comprises a first nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) nucleotide sequence, a second nucleotide sequence comprising an open reading frame (ORF) comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell, a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell. The method comprises introducing the at least one vector into an insect cell and maintaining the insect cell under conditions such that AAV is produced.
In accordance with another aspect of the invention, another method of producing AAV in an insect cell is provided. The method comprises providing an insect cell comprising (a) a first nucleotide sequence comprising at least one AAV ITR nucleotide sequence, a second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2 and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell, a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and, optionally, (b) at least one insect cell-compatible vector. At least one of the first, second, third and fourth nucleotide sequences is/are stably integrated in the insect cell and the at least one insect cell-compatible vector, when present, comprises the remainder of the first, second, third and fourth nucleotide sequences which is/are not stably integrated in the insect cell. The insect cell is maintained under conditions such that AAV is produced.
In accordance with a further aspect of the invention, insect cell-compatible vectors are provided. One vector comprises a nucleotide sequence encoding Rep78 or Rep68 operably linked to a modified early 1 gene (IE-1) promoter from Orgyia pseudotsugata (xcex94IE-1) and a Kozak-like expression control sequence. Another vector comprises an ORF comprising 3 nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence comprising a nine nucleotide sequence of SEQ ID NO:4 or a sequence substantially homologous to SEQ. ID NO: 4, upstream of an initiation codon of the nucleotide sequence encoding AAV VP1 capsid protein, and a C at nucleotide position 2 of the nucleotide sequence encoding AAV VP1 capsid protein.
In yet another aspect of the invention, an insect cell is provided. The insect cell comprises a first nucleotide sequence comprising at least one AAV ITR nucleotide sequence, a second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell, a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell.
In accordance with another aspect, yet another method of producing AAV in an insect cell is provided. The method comprises providing at least one insect cell-compatible vector comprising a first nucleotide sequence comprising at least one chimeric ITR nucleotide sequence, the ITR nucleotide sequence comprising an AAV backbone and a specific binding and a nicking site of a Rep protein from a parvovirus other than AAV, a second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell, a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and a fourth nucleotide sequence comprising a nucleotide sequence encoding a parvoviral Rep protein that can specifically bind and nick the site in the ITR nucleotide sequence within the first nucleotide sequence, operably linked to at least one expression control sequence for expression in an insect cell. The method further comprises introducing the at least one insect cell-compatible vector into an insect cell, and maintaining the insect cell under conditions such that AAV is produced.
The invention also provides a method of producing a parvoviral genome in an insect cell by introducing at least one insect cell-compatible vector into the insect cell and thereafter maintaining the insect cell under conditions such that a parvoviral genome is produced in the cell. The one or more insect cell-compatible vectors used in the method collectively include a first nucleotide sequence comprising at least one parvoviral ITR, a second nucleotide sequence comprising an AAV Rep52 or Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and a third nucleotide sequence comprising an AAV Rep78 or Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell.